The present invention relates to transgenic plants having an increased free amino acid content, and a method of producing them. In particular, the present invention relates to transgenic plants containing at least one of asparagine, aspartic acid, serine, threonine, alanine, histidine and glutamic acid accumulated in a large amount, and a method of producing them.
The technique of transforming a plant by introducing a specified gene thereinto, reported for the first time in the world, in the study where it was achieve by introducing a gene into tobacco with Agrobacterium tumefaciens, a soil microorganism. Thereafter, many products having useful agricultural characters were produced, and it was also tried to let plants produce useful components. A plant breeding method by such a recombinant DNA technique is considered to be hopeful in place of the ordinary, traditional technique of developing varieties of plants. In this field, improvement in the characteristics of plants concerning nitrogen assimilation is also being studied. The study of amino acids is particularly prospering because they are important ingredients of particularly fruits, root crops and seeds and also they exert a great influence on the tastes of them.
Reports on the biosynthesis of amino acids include, for example, a report that free lysine content of tobacco was increased to 200 times as high content by introduction of E. coli DHDPS gene into tobacco (U.S. Pat. No. 5,258,300, Molecular Genetics Res. & Development); a report that free lysine content was increased by introduction of AK gene (EP 485970, WO 9319190); a report that asparagine content was increased to 100 times as high content by introduction of AS gene into tobacco; and a report that tryptophan content was increased to 90 times as high content by introduction of an anthranilic acid-synthesizing enzyme into a rice plant (WO 9726366, DEKALB Genetic Corp). The plants in which a gene is to be induced are not limited to model plants such as tobacco and Arabidopsis thaliana but plants which produce fruits such as tomato are also used. For example, as for tomatoes, a transformant thereof was obtained by Agrobacterium co-cultivation method in 1986 [S. McCormick, J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Fraley, Plant Cell Reports, 5, 81-84 (1986); Y. S. Chyi, R. A. Jorgenson, D. Goldstern, S. D. Tarksley and F. Loaiza-Figueroe, Mol. Gen. Genet., 204, 64-69 (1986)]. Since then, investigations were made for the improvement of the transgenic plant lines. Various genes relating to the biosynthesis of amino acids and nitrogen assimilation other than those described above are also known. They include asparaginase and GOGAT, and the base sequences of them were also reported.
Glutamic acid which is one of α-amino acids is widely distributed in proteins. It is generally known that a tasty component of tomatoes used as a seasoning and also a tasty component of fermentation products of soybeans (such as soy sauce and fermented soy paste) are glutamic acid. It is also known that glutamic acid is synthesized in the first step of nitrogen metabolism in higher plants. It is also known that glutamine and asparagine formed from glutamic acid are distributed to tissues through phloems and used for the synthesis of other amino acids and proteins. It was reported that in plants, glutamic acid is contained in a high concentration in phloems through which photosynthesis products such as sucrose and amino acids are transported [Mitsuo Chino et al., “Shokubutsu Eiyo/Hiryogaku” p. 125 (1993)]. As for examples of cases wherein glutamic acid is contained in a high concentration in edible parts of plants, it is known that about 0.25 g/100 gf. w. of glutamic acid is contained in tomato fruits [“Tokimeki” No. 2, Nippon Shokuhin Kogyo Gakkaishi, Vol. 39, pp. 64-67 (1992)]. However, glutamic acid of a high concentration cannot be easily accumulated in plant bodies because it is a starting material for amino group-donors and also it is metabolized in various biosynthetic pathways as described above even though the biosynthesizing capacity of the source organs can be improved. As far as the applicant knows, it has never been succeeded to remarkably increase glutamic acid concentration in edible parts of plants by either mated breeding or gene manipulation.
In the first step of the assimilation of inorganic nitrogen into an organic substance, ammonia is incorporated into glutamic acid for mainly forming glutamine. This process is catalyzed by glutamine synthetase (GS). Then glutamine is reacted with α-ketoglutaric acid in the presence of glutamate synthase (GOGAT) to form two molecules of glutamic acid. This GS/GOGAT cycle is considered to be the main pathway of nitrogen assimilation in plants [Miflin and Lea, Phytochemistry 15; 873-885 (1976)]. On the other hand, it is also known that the ammonia assimilation proceeds also through a metabolic pathway other than the pathway wherein ammonia is incorporated in the presence of GS catalyst [Knight and Langston-Unkefer, Science, 241: 951-954 (1988)]. Namely, in this metabolic pathway, ammonia is incorporated into α-ketoglutaric acid for forming glutamic acid. This process is catalyzed by glutamate dehydrogenase (GDH). However, plant GDH has a high Km value for ammonia. The role of this pathway under normal growing conditions has not yet been elucidated enough because ammonia is toxic and the concentration of intracellular ammonia is usually low. A researcher reported that this pathway contributes to the nitrogen assimilation when ammonium concentration in the cells is increased over a normal level (the above-described literature of Knight and Langston-Unkefer).
In plants, glutamate dehydrogenase (GDH) catalyzes a reversible reaction of taking ammonia into α-ketoglutaric acid to form glutamic acid and, on the contrary, deamination from glutamic acid to form α-ketoglutaric acid. It is considered that the former reaction occurs when the amount of ammonia is large, and the latter reaction occurs when nitrogen content is high [Robinson et al., Plant Physiol. 95; 809-816 (1991): and Robinson et al., Plant Physiol. 98; 1190-1195 (1992)]. The directionality of this enzyme is not fixed unlike an enzyme GDH-A which acts in microorganisms to synthesize glutamic acid or an enzyme GDH-B which acts on them to decompose it. In plants, it is considered that there are two kinds of such enzymes, i.e. NADP-depending GDH which functions in chloroplast and NAD-dependent GDH which functions in mitochondria. Since GDH has a high Km value for ammonia and it is highly related to the ammonia level in the photorespiration, it is supposed that NAD-dependent GDH localized in mitochondria has an important role in the assimilation of ammonia [Srivastava and Singh R P, Phytochemistry, 26; 597-610 (1987).
It is known that plant GDH comprises a hexamer composed of two different kinds of polypeptides (α-subunits and β-subunits) linked with each other at random and also that there are seven isozyme patterns depending on the degree of the linkage. After investigations wherein grapevine calli were used, the following facts were reported: When calli cultured in a medium containing a nitrate and glutamic acid were subjected to the electrophoresis, an isozyme comprising β-subunits was increased on the cathodic side. On the contrary, when calli cultured in a medium containing ammonia and glutamine were subjected to the electrophoresis, an isozyme comprising α-subunits was increased on the anodic side. Further, when the calli were transferred from the nitrate medium into the ammonia medium, GDH activity was increased to 3 times as high activity (α-subunits were increased to 4-times and β-subunits were decreased). Thus, the activity moved from the cathodic side to the anodic side [Loulakakis and Poubelakis—Angelakis, Plant Physiol. 97; 104-1111 (1996)]. According to this report, α-subunits were considered to play an important role in the assimilation of ammonia.
Sakakibara et al. [Plant Cell Physiol., 33; 1193-1198 ] isolated GDH genes from two isozyme bands on the cathodic side in seven isozyme bands in corn roots at the first time in the field of plants in 1995. Thereafter, GDH genes were isolated from grapevine [Syntichaki et al., Gene 168: 87-92 (1996)], Arabidopsis [Melo-Olivera et al., Proc. Natl. Acad. Sci., USA 93; 4718-4723 (1996)] and tomato [Purnell et al., Gene 186; 249-254 (1997)]. In particular, the genes isolated from grapevine calli were isolated from an isozyme expressed in ammonia-treated cells, and they are considered to be genes encoding α-subunits. All the genes contain a transit peptide which is functional in mitochondria. GDH genes of corns and tomatoes are expressed in a large amount in their roots, while those of Arabidopsis are expressed in the leaves and flowers. It was suggested that only one copy of gene is present in tomato, while two or more genes are present in corn, Arabidopsis and grapevine. This fact suggests that also in plants, the constitution and function of genes are different and complicated.
Transgenic plants in which said GDH gene was introduced were also produced. It was reported that when glutamate dehydrogenase GDH (NADP-GDH) gene from Escherichia coli was introduced into tobacco and corn for the purpose of imparting resistance to phosphinothricin used as a herbicide, glutamic acid content of the roots of them was increased to 1.3 to 1.4 times as high [Lightfoot David et al, CA 2180786 (1998)]. Namely, according to this report, glutamic acid content of tobacco roots was increased from 14.7 mg/100 gf.w. to 20.6 mg/100 gf. w., and that of corn roots was increased from 16.2 mg/100 gf. w. to 19.1 mg/100 gf w. Although there are other reports on the use of GDH gene, no example is given therein [WO 9509911, α,β-subunits from chlorella (WO 9712983)]. In addition, no analytical value of amino acids of glutamic acid group was given therein.